Fanconi anemia (FA) is an autosomal recessive disorder characterized clinically by progressive pancytopenia, diverse developmental abnormalities and a predisposition to malignancy. Although the molecular basis for the syndrome is unknown, hypersensitivity of cells to the clastogenic and cytotoxic effects of DNA crosslinking agents such as diepoxybutane and mitomycin-C can be used as a cellular marker for the disorder. In this project, genetic heterogeneity in FA will be characterized by performing complementation analysis in euploid somatic cell hybrids constructed by fusing cells from different FA patients, using hybridoma technology. Persistence of the FA cellular phenotype will indicate a lack of complementation, while correction of the defect in hybrid cells will indicate that the parental lines belong to different complementation groups. Hybrids will also be constructed between FA homozygous, FA heterozygous and normal cells in various combinations. These experiments will facilitate study of gene dosage effects, and may be useful for heterozygote detection. The sensitivity of FA homozygous, FA heterozygous, normal control and hybrid cells to exposure to various oxygen tensions in the presence of DNA crosslinking agents will be assayed as a potential method for discrimination of heterozygotes. The hypersensitivity of FA cells to DNA crosslinking agents will also be used as a selectable marker in gene transfer experiments. An attempt will be made to isolate resistant clones and "rescue" the transforming sequences, using a cDNA library package in a bacteriophage lambda vector that has a high transformation efficiency. If the transforming sequences can be isolated and cloned, their chromosomal location and nucleotide sequence will be determined, and the molecular basis for the defect in various FA patients characterized. As an alternative approach to mapping the FA gene, linkage analysis of DNA markers will be performed to localize tHe FA gene(s) to a specific chromosome(s). A major resource and unique feature of this proposal is our access to a large number of FA patients exhibiting the full spectrum of its diverse features, through the International Fanconi Anemia Registry (IFAR) maintained by us at The Rockefeller University.